Pathway Assignments

The assignment – Annotating Pathways KEGG Pathway Database

The assignment – Annotating Pathways KEGG Pathway Database All teams will be assigned a different pathway. This one is provided only as an example. Energy Metabolism Nitrogen metabolism

Closely related to P. limnophilus The green boxes correspond to genes found in R. baltica Which genes are found in P. limnophilus? Hypothesis: If P. limnophilus uses nitrogen as NRG source, then it likely will have the same genes as R. baltica.

Search by EC number

Field M10 of the Roche Applied Science “Biochemical Pathways” wall chart

“Click” What do you do with assigned gene? 1- Go to KEGG Pathway Database 2- Click on “green box” corresponding to the gene that was assigned to you.

Amino acid sequence “Click” for FASTA file 3- Obtain amino acid sequence (FASTA format) from KEGG for assigned R. baltica gene

FASTA format: Description line preceded by greater than sign Amino acid sequence for protein encoded by corresponding gene NOTE: Nucleotide sequences also can be depicted in FASTA format 4- “COPY/PASTE” entire entry (including description line) into text editing program (i.e., Notepad, Wordpad) for use in subsequent database searches

5- In the text editor (Notepad), save the file containing the amino acid sequence in FASTA format as plain text file (.txt) 6- Return to imgACT

9- Right click on img/edu icon and open Organism Details page for Planctomyces limnophilus DSM 3776 “Click” 10- Click “Find Genes” tab “Click” Get used to right-click then open in new tab

11- Click [BLAST] from menu in “Find Genes” tab “Click” 12- Retrieve amino acid sequence for R. baltica gene. Copy/paste into query box. Select blastp program with E-value cutoff of 1e -2 and P. limnophilus genome as the database to be searched. Then hit.

Referred to as a “Reciprocal BLAST” search in instructions

13- Results of BLAST search will be displayed in format similar to what is generated with NCBI database Inspect “hit” list

14- Select best “hit” and add to “Gene Cart” Scroll down

Note Gene Object ID (OID) 14- Return to imgACT

- imgACT “Student Teams” will be created by the instructor once all students have registered for course - Find your course “Home” page on imgACT Select MIMG 103L, Winter 2010

- View your team(s) by clicking on Student Team name “Click” Joe Jenny Jess Jeffrey Josie

- View your Practice Gene by clicking link under “Gene Annotation Assignments” “Click”

- View your team ASSIGNMENT by clicking link under “Pathway/Structure Assignments” “Click”

You should see the “Pathway/Structure Report” page Notice team members with addresses (Your TA & instructors also will be listed here)

As a TEAM: set up your team notebook page “Click”

Add pathway & gene information for reference organism Insert KEGG map: - return to KEGG database - save KEGG map as.gif or.png

Must be in EDIT mode to insert image Place cursor where you want to insert image “Click” NOTE: Upload entire KEGG map. Only crop the image if assigned structure/complex or pathway is portion of full KEGG map “Click”

Result after image inserted and comments added to box:

Each person on the team should enter the information for the Reference Organism & Gene used for their Reciprocal BLAST search: 1.This step requires that you create additional boxes and headings. While in EDIT mode, simply copy/paste then modify the text. 2.Include full name of reference organism. 3.Include amino acid sequence used for the reciprocal BLAST search (copy/paste from Notepad).

Press SAVE button so you don’t lose your work. “CLICK” NOTE: Additional information for MetaCyc will be entered on the page later. IMPORTANT NOTES!! Only ONE person at a time from each team may modify the team notebook; otherwise you will delete your teammate’s work! Skip MetaCyc section – you may add this later in the quarter.

Return to “Pathway/Structure Report” in imgACT “Click” Individually add steps or components to your pathway or structure

Enter the “Step name” and “Gene Object ID”. Then press. Your TA may have suggestions for “step name” (or component name for cell structures) if you are not sure what to call it. The Gene Object ID corresponds to top reciprocal BLAST hit in P. limnophilus genome.

“Pathway/Structure Report” page should be updated Indicates step creation was successful

Log out of lab notebook

Sign out of imgACT “CLICK” Close all tabs and windows before exiting the browser